CN103328955B - Utilize the light analysis method and light analytical equipment of the detection of the light from single incandescnet particle - Google Patents
Utilize the light analysis method and light analytical equipment of the detection of the light from single incandescnet particle Download PDFInfo
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- G—PHYSICS
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- G01J1/00—Photometry, e.g. photographic exposure meter
- G01J1/42—Photometry, e.g. photographic exposure meter using electric radiation detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
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- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
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Abstract
In the scanning numerator counts method of the photo measure of confocal microscope or multiphoton microscope has been used, can the deviation of result be suppressed small independent of the concentration of incandescnet particle and optimize time of measuring.Light of the detection from incandescnet particle and the technology analyzed of the present invention repeat on one side by change the light path of microscopical optical system make optical system photo detection area position in sample solution the mobile intensity for detecting the light from photo detection area on one side and individually detect incandescnet particle light signal processing untill the number of the signal from incandescnet particle reaches pre-determined number, the time required for pre-determined number is reached according to the number of the signal from incandescnet particle to determine the concentration of the incandescnet particle in sample solution.
Description
Technical field
The present invention relates to a kind of smooth analysis method and light analytical equipment, confocal microscope or multiphoton microscope can be used
Optical system etc. can detect tiny area in solution light optical system, detection is molten from being dispersed or dissolved in
Atom, molecule or their condensate (hereinafter called them " particle "), such as protein, peptide, nucleic acid, lipid in liquid,
Object or the abiology grain of the particle shapes such as organism molecule, virus, the cells such as sugar chain, amino acid or their condensate
The light of son, to obtain the information useful when analyzing or parsing their state (interaction, with reference to/dissociated state etc.), more
In detail, be related to it is a kind of can use optical system as described above individually detection the light from single luminous particle go forward side by side
The method and apparatus of the various light analyses of row.In addition, in this manual, luminous particle (hereinafter referred to as " incandescnet particle ") can be with
It is its own luminous particle or addition of the particle of arbitrary luminous mark or luminescence probe, the light sent from incandescnet particle
Can be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light etc..
Background technology
Due to the development of optical measurement techniques in recent years, using confocal microscope optical system and photon can also be carried out
The optical detection technique of the hypersensitivity of (singl e photon detection) is counted, single photon or fluorescence single molecules level can be detected/measure
Faint light.Therefore, it is proposed to which the various e measurement technologies using such faint lights are to the characteristic of organism molecule etc., intermolecular
The device or method that interaction or binding/dissociation reaction are detected.For example, in fluorescence correlation spectroscopy analysis
(Fluorescence Correlation Spectroscopy：FCS.Referring for example to patent document 1-3, non-patent literature 1-3)
In, using the optical system and photon counting technique of laser scanning confocal microscopy, measure the Microcell in the sample solution that comes in and goes out
Fluorescence molecule in domain (being referred to as focus area-confocal district's groups that microscopical laser converges to knit) or point by fluorescence labelling
The fluorescence intensity of sub (fluorescence molecule etc.), according to determined by the value of the auto-correlation function of the fluorescence intensity obtained as measurement
The average value of the mean residence time (translation diffusion time) of fluorescence molecule in tiny area etc. and the number of stranded molecule, is obtained
Take the information of speed or size, the concentration of the motion of fluorescence molecule etc. etc, or detection molecules construction or size change, point
The various phenomenons of the binding/dissociation reaction of son or scattered/polymerization etc.In addition, in fluorescence intensity distributional analysis
(Fluorescence-Intensity Distribution Analysis：FIDA.Such as patent document 4, non-patent literature
4), photon counting histogram (Photon Counting Histogram：PCH.Such as patent document 5) in, generate same with FCS
The confocal district's groups of discrepancy measured knit the histogram of the fluorescence intensity of interior fluorescence molecule etc. sample, make the model formation of statistics
The histogrammic distribution is fitted, the average value of the intrinsic brightness of fluorescence molecule etc. is thus estimated and is trapped in confocal district's groups and knit interior
Molecule number average value, according to these information estimate the construction of molecule or the change of size, with reference to/dissociated state, point
Dissipate/polymerization state etc..In addition, in addition, in patent document 6,7, it is proposed that according to the optical system using confocal microscope
The method that the time of the fluorescence signal for the sample solution that unified test is measured detects fluorescent substance through coming.Patent document 8 is proposed
A kind of signal operation treatment technology, it is used for using photon counting technique measurement from the fluorescent particle for circulating in flow cytometer
The faint light of son or the fluorescent micro par-ticles being fixed on substrate, to detect the presence of the fluorescent micro par-ticles in fluid or on substrate.
Particularly, optical system and the photon counting technique progress using confocal microscope have been used according to FCS, FIDA etc.
The method of the technology of the fluorescence measurement of tiny area, sample required for measuring with can be than before extremely low concentration and
Denier (amount used in one-shot measurement is at most tens μ L or so), time of measuring also significantly shortens (in one-shot measurement
The repeated multiple times measurement for carrying out level time second).Thus, expect these technologies particularly in the research and development to medical science, biology
In the case that the rare or expensive sample being commonly used in field is analyzed, or clinical diagnosis in disease, physiologically active
In the case that the detection body number such as screening of material is more, as can inexpensively or promptly be held compared with former biochemical method
Row experiment or the powerful tools checked.
Patent document 1：Japanese Unexamined Patent Publication 2005-098876
Patent document 2：Japanese Unexamined Patent Publication 2008-292371
Patent document 3：Japanese Unexamined Patent Publication 2009-281831
Patent document 4：Te Xu the 4023523rd
Patent document 5：International Publication 2008-080417
Patent document 6：Japanese Unexamined Patent Publication 2007-20565
Patent document 7：Japanese Unexamined Patent Publication 2008-116440
Patent document 8：Japanese Unexamined Patent Publication 4-337446 publications
Non-patent literature 1：Golden city political affairs filial piety, Vol.44, No.9,1431-1438 pages of protein nuclease 1999 years
Non-patent literature 2：F.J.Meyer-Alms, fluorescence correlation spectroscopy (Fluorescence Correlation
Spectroscopy), R.Rigler volumes, Springer, Berlin, 2000,204-224 pages
Non-patent literature 3：Plus rattan then son and other 4, medical genetics, Vol.6, No.2,271-277 page
Non-patent literature 4：Ka Sike (Kask) and other 3, American science institute summary, volume 96,13756- in 1999
Page 13761 (P.Kask, K.Palo, D.Ullmann, K.Gall PNAS 96,13756-13761 (1999))
The content of the invention
Problems to be solved by the invention
The optical system of confocal microscope and the optical analysis of photon counting technique have been used in above-mentioned FCS, FIDA etc.
In technology, the measured light just sent from fluorescence unimolecule or polymolecular, but in the analysis of the light, execution is sequentially surveyed
The computing of the auto-correlation function of the fluorescence intensity data measured or the calculating of fluctuation to the fluorescence intensity of histogram-fitting etc
Deng statistical processing, not individually with reference to or light of the analysis from each fluorescence molecule etc. signal.That is, in these light point
In analysis technology, the signal statistics of the light from multiple fluorescence molecules etc. are handled, detects and unites for fluorescence molecule etc.
Count the characteristic of evenness.Thus, it is statistically significant as a result, it is desirable to sample solution in order to be obtained in these light analysis technologies
In the concentration or number density as the fluorescence molecule of object of observation etc. in the state of the equilibrium be second level length once survey
The level that fluorescence molecule of number of processing of statistics etc. comes in and goes out in tiny area can be carried out in the amount time, is preferably existed
All the time there is the level of the fluorescence molecule of one or so etc. in tiny area.In fact, the volume that confocal district's groups are knitted is left for 1fL
The right side, therefore, the concentration of fluorescence molecule of the sample solution used in above-mentioned light analysis technology etc. be typically 1nM or so or
More than it, when being significantly less than 1nM, produce the time that fluorescence molecule etc. is not present in confocal district's groups are knitted and can not be counted
Upper significant analysis result.On the other hand, in the detection method of the fluorescence molecule described in patent document 6~8 etc., do not wrap
The statistical calculation process of the fluctuation of fluorescence intensity is included, the fluorescence molecule even if in sample solution etc., which is less than 1nM, can also examine
Fluorescence molecule etc. is surveyed, but can not quantitatively calculate the concentration or number density of the fluorescence molecule of random motion in the solution etc..
Therefore, present applicant is in Japanese Patent Application 2010-044714 and PCT/JP2011/53481, it is proposed that base
In the light analysis technology of following new principle, the concentration or number density of the incandescnet particle as object of observation can be quantitatively observed
Less than luminous in the sample solution of the level handled using the light analysis technology comprising statistical processing such as FCS, FIDA
The state or characteristic of particle.It is same with FCS, FIDA etc. if be clearly described in the new light analysis technology
Ground, the light of the tiny area in solution can be detected in optical system using confocal microscope or multiphoton microscope etc.
Optical system in the case of, while making the position of the tiny area (hereinafter referred to as " photo detection area ") of the detection zone as light
Put and moved in sample solution, i.e. while by photo detection area to being scanned in sample solution, while in photo detection area
The light sent from the incandescnet particle is detected during the scattered and incandescnet particle of random motion included in sample solution, thereby, it is possible to
Incandescnet particle in sample solution is one by one detected individually, to obtain in counting with incandescnet particle, sample solution
The relevant information of the concentration or number density of incandescnet particle.Numerator counts (hereinafter referred to as " are scanned according to the new light analysis technology
Method "), in the same manner as the light analysis technology such as FCS, FIDA, the sample required for measuring can be micro (such as tens μ L
Left and right), in addition, time of measuring is short, and compared with the situation of the light analysis technologies such as FCS, FIDA, lower concentration can be detected
Or the presence of the incandescnet particle of number density, and quantitatively detect its concentration, number density or other characteristics.
In addition, incandescnet particle in by above-mentioned " scanning numerator counts method " individually test sample solution is determined
In the case of concentration or other characteristics, preferred pair reaches that the incandescnet particle of the number of the precision required by its result is detected.
For example when determining the concentration of incandescnet particle in sample solution by scanning numerator counts method, following handle is carried out：To indivedual
The number of the incandescnet particle detected is counted, by the count number divided by time of measuring (time for performing the detection of light)
Photo detection area the cumulative volume by region.In this case, incandescnet particle is randomly dispersed in sample solution, because
This needs the count number of incandescnet particle to reach enough numbers so that its deviation diminishes to accurately determine concentration.Sample
The concentration of incandescnet particle in this solution is higher, and the count number of incandescnet particle is more within the short time, therefore incandescnet particle
The higher time of measuring detected there is provided the incandescnet particle of the number of required precision of concentration becomes shorter.In other words, logical
In the case of crossing concentration or other characteristics that " scanning numerator counts method " determines incandescnet particle in sample solution, dependent on luminous
The concentration of particle and required time of measuring is different.
However, in the case where the concentration as the incandescnet particle of object of observation in sample solution is unknown, there is provided want
Time of measuring required for the incandescnet particle detection of the number for the precision asked is unclear, therefore time of measuring is set to
The detection of the incandescnet particle of the number of precision required by can also being provided in the case that the concentration of incandescnet particle is low.At this
In the case of kind, the high sample solution of concentration on incandescnet particle there is a situation where to cause time of measuring with exceeding needs elongated.
In addition, when the concentration not based on incandescnet particle carries out the detection of incandescnet particle with identical time of measuring, it may occur however that with
Lower situation：In the case where the concentration of incandescnet particle is high, deviation as a result is small, but in the low situation of the concentration of incandescnet particle
Under, deviation as a result becomes big.
So, a problem of the invention is that offer is a kind of to the greatest extent can may be used in above-mentioned " scanning numerator counts method "
End reaches the new method and device of the detection of the incandescnet particle of the number of the precision required by result in a period of energy is short.
In addition, another problem of the present invention be to provide it is a kind of in above-mentioned " scanning numerator counts method " independent of work
The new method and device for for the concentration of the incandescnet particle of object of observation the deviation of result being suppressed small.
Also, another problem of the present invention be to provide it is a kind of being capable of basis in above-mentioned " scanning numerator counts method "
Make the new method and device of time of measuring optimization as the concentration of the incandescnet particle of object of observation.
The solution used to solve the problem
According to the present invention, above-mentioned problem is reached by following method, this method is to use confocal microscope or many
The optical system of photon microscope comes the light of the incandescnet particle of scattered and random motion and progress in comfortable sample solution to detect
The method of analysis, it is characterised in that comprise the following steps：Photo detection area moving step, by the light path for changing optical system
Moved come the position for the photo detection area for making optical system in sample solution；Light detecting step, while making photo detection area
Position is mobile in sample solution while light of the detection from photo detection area；And incandescnet particle detecting step, according to being examined
The light measured, individually detects the signal from each incandescnet particle, wherein, repeat above three step spontaneous until coming
Untill the number of the signal of light particle reaches pre-determined number, reached in advance according to the number of the signal from incandescnet particle
Time required for the number of decision, to determine the concentration of the incandescnet particle in sample solution.
In the structure of the invention, " the scattered and random motion in sample solution " incandescnet particle refers in sample
Atom, molecule or their condensate for being dispersed or dissolved in solution etc. send the particle of light, as long as substrate etc. is not fixed on
Particle that is upper and freely carrying out Brownian movement in the solution, then can be arbitrary particle.Incandescnet particle is typically fluorescence
Property particle is but it is also possible to be the particle that light is sent by phosphorescence, chemiluminescence, bioluminescence, light scattering etc..Confocal microscope
Or " photo detection area " of the optical system of multiphoton microscope refers to the tiny area of detection light in these microscopes, from thing
In the case that mirror provides illumination light, the region converged to equivalent to the illumination light is (in confocal microscope, particular according to object lens
Position relationship between pin hole is determined.Incandescnet particle be no illumination light and it is luminous in the case of, for example pass through chemistry
It is luminous or in the case that bioluminescence carrys out luminous particle, illumination light is not needed in microscope.).In addition, in this specification
In, in the case where being referred to as " signal ", as long as no being particularly limited to, refer to the signal for representing the light from incandescnet particle.
As appreciated from the above, in the scanning numerator counts method as the basic structure of the present invention, first, on one side
Making the position of photo detection area be moved in sample solution, i.e. while by photo detection area to being swept in sample solution
Retouch, carry out the detection of light successively on one side.So, it can expect to contain the photogenic granules of random motion in mobile photo detection area
The period of the day from 11 p.m. to 1 a.m detects the light from incandescnet particle, thus detects the presence of an incandescnet particle.Thus, in the light detected successively
In individually detect the signal of the light from incandescnet particle, the presence of particle is thus one by one individually detected successively, can be obtained
The various information for taking the state with particle in solution relevant.The concentration of incandescnet particle in not based on sample solution and at certain
The a series of step, i.e. photo detection area moving step, light detecting step are performed in fixed time of measuring and is lighted
In the case of detection of particles step, as described above according to the difference of the concentration of incandescnet particle incandescnet particle detection number
Deviation is different, and when the concentration of incandescnet particle is high, it is possible to time of measuring is elongated more than needing, in the dense of incandescnet particle
When spending low, it is possible to the detection of the incandescnet particle for the number for reaching the precision required by measurement can not be reached.
Therefore, in the method for the invention, be not performed in the time of measuring of certain fixation photo detection area moving step,
Light detecting step and incandescnet particle detecting step, but repeat above step as described above until from incandescnet particle
Signal number reach pre-determined number untill.Then, gauge check is to the incandescnet particle of pre-determined number
Only required time, the time required for pre-determined number is reached according to the number of the signal from the incandescnet particle
To determine the concentration of the incandescnet particle in sample solution.According to the structure, the concentration of incandescnet particle is higher, the inspection of incandescnet particle
Survey number and pre-determined number is reached within the shorter time, therefore expect contracting for the high sample solution of the concentration of incandescnet particle
Short time of measuring, the low sample solution of concentration for incandescnet particle spends time enough to perform measurement.That is, according to above-mentioned knot
Structure, can optimize time of measuring according to the concentration of incandescnet particle.In addition, if pre-determined number is set as in advance
Reach the number of the precision required by result, then can be pre- prerequisite by the detection of the low sample solution of the concentration for incandescnet particle
Time required for the incandescnet particle of fixed number or the deviation based on its derived arbitrary result suppress small, so as to meet
As a result precision.
In the structure of above-mentioned method of the invention, the concentration of incandescnet particle is reflected as the signal from incandescnet particle
Number reach time required for pre-determined number, it will therefore be appreciated that can be according to from the incandescnet particle
The number of signal reaches the time required for pre-determined number to determine the concentration of incandescnet particle.Specifically, it can make
Reach the arbitrary function of time required for pre-determined number for the number of the signal of spontaneous light particle and calculate hair
The concentration of light particle.For example according to the detection number (that is, pre-determined number) of incandescnet particle and the letter from incandescnet particle
Number the number detection speed of incandescnet particle that reaches the time required for pre-determined number and determine (time per unit
Detect number) it is proportional to the concentration of incandescnet particle, therefore be advantageously used.
In addition, as understood from the explanation on the column of embodiment one described later, in photo detection area moving step and
The light from sample solution is detected in light detecting step and hair is detected in incandescnet particle detecting step according to resulting data
, it is necessary to carry out removing noise from resulting data determining to light in the above-mentioned a series of step of the presence of light particle
The processing of the signal of particle.The processing can be reached by following method, i.e., carry out light in the time of measuring of certain fixation
Detection when, after the detection of the light in the time of measuring of the fixation is completed, resulting data are unified to be analyzed.
However, performing feelings of the detection of incandescnet particle untill the number of the signal from incandescnet particle reaches pre-determined number
, it is necessary to detect the signal from incandescnet particle in the midway of photo detection area moving step and light detecting step under condition.Therefore, exist
In the structure of above-mentioned method of the invention, it can reach that pre-determined number is in the number of the signal from incandescnet particle
Repeat photo detection area moving step, light detecting step and photogenic granules by each predetermined time interval in a period of only
Sub- detecting step.For the predetermined time interval, can be it is fixed, can also the signal from incandescnet particle
In a period of number is reached untill pre-determined number, it is modified according to the detection number of the incandescnet particle untill at that time.It is special
Be not in the latter case, can after measurement is actually started incandescnet particle in sample solution detected status come
Adjustment repeats predetermined time interval during above-mentioned a series of step, can further improve the optimization of time of measuring.
Also, can be in the signal from incandescnet particle as an embodiment of above-mentioned method of the invention
Number reach untill pre-determined number in a period of, execution estimated according to the detection number of the incandescnet particle untill at that time
The step of number of signal of the meter from incandescnet particle reaches the time required for pre-determined number.Specifically, for example
After detection starts, the detection number of the elapsed time after being started according to detection and the incandescnet particle untill at that time is come really
Determine the detection speed of incandescnet particle, the number for estimating the signal from incandescnet particle according to the detection speed of the incandescnet particle reaches
Time to required for pre-determined number.According to the structure, it can predict when time of measuring terminates, for experimenter
For be easily information.
In addition, the position on the photo detection area in above-mentioned structure of the invention is moved, can be according to incandescnet particle
Characteristic or sample solution in concentration suitably change photo detection area in sample solution position translational speed.Particularly
When the translational speed of photo detection area accelerates, the light quantity obtained from an incandescnet particle is reduced, it is therefore preferred to suitably
The translational speed of photo detection area is changed to measure the light from an incandescnet particle in high precision or in high sensitivity.
Also, the movement of the position on above-mentioned photo detection area, preferably by the light detection zone in sample solution
The translational speed of the position in domain is set to the diffusion translational speed than incandescnet particle (as being averaged for the particle caused by Brownian movement
Translational speed) it is high.As described above, in the method for the invention, one included from photo detection area is lighted
The light that particle is sent is detected, detects incandescnet particle individually.However, incandescnet particle due to carrying out Brownian movement in the solution
And randomly move, in the case of the photo detection area that repeatedly comes in and goes out, it is possible to from an incandescnet particle repeated detection to (expression
The presence of the incandescnet particle) signal, it is difficult to the signal detected and the presence of an incandescnet particle is mapped.Therefore,
As described above, the translational speed of photo detection area is set to higher than the diffusion translational speed of incandescnet particle, thus enabled that
One incandescnet particle one signal of correspondence.In addition, diffusion translational speed changes because of the difference of incandescnet particle, therefore as above institute
State, the translational speed of photo detection area is preferably suitably changed according to the characteristic of incandescnet particle.
The change of light path of optical system can be carried out by arbitrary mode to move the position of photo detection area.For example
The galvanomirror (Galvano-mirror) employed in laser scanning type light microscope can be used to change light path, to change
The position of photo detection area.The mobile route of the position of photo detection area can be arbitrarily set, for example can be from circular, ellipse
Selected in shape, rectangle, straight line and curve.In addition, in the present invention, being configured to change the light path of optical system to make light
The position movement of detection zone, thus the movement of photo detection area is rapid, and does not substantially occur machinery in sample solution
Incandescnet particle in vibration, hydrokinetic effect, therefore sample solution will not be influenceed by mechanical function and (can not had
Have pseudomorphism (artifact) state and) it is stable in the state of carry out the measurement of light (such as in the situation for flowing sample
Under, it is difficult to the same flow velocity is assigned all the time, and apparatus structure is complicated, and required sample size is significantly increased in addition, and by
In the hydrokinetic effect caused by flowing, incandescnet particle or other materials in the solution is likely to occur modification or changed
Property.).Also, it is not necessary to make sample solution circulate structure, therefore, it is possible in the same manner as FCS etc. situation with it is micro (1~it is several
Ten μ L or so) sample solution measure and analyze.
Above-mentioned method of the invention is realized by new light analytical equipment, the light analytical equipment can be while examine light
The position for surveying region is moved in sample solution, while detecting the light of each incandescnet particle.So, in another side of the present invention
In formula, reach above-mentioned problem of the invention by following light analytical equipment, the light analytical equipment using confocal microscope or
The optical system detection of multiphoton microscope comes the light of the scattered and incandescnet particle of random motion in comfortable sample solution, the light point
Analysis apparatus is characterised by, including：Photo detection area move portion, it makes light by changing the light path of microscopical optical system
Moved in sample solution the position of the photo detection area of system；Optical detection part, it detects the light from photo detection area；With
And signal processing part, it is to from making the position of photo detection area be moved in sample solution while being detected by optical detection part
The signal of each incandescnet particle gone out individually detected, wherein, repeat the optics using photo detection area move portion
The movement of the position of the photo detection area of system, the detection and utilization to the light from photo detection area using optical detection part
The detection to the signal from incandescnet particle of signal processing part, until detected by signal processing part from incandescnet particle
Untill the number of signal reaches pre-determined number, pre-determined is reached according to the number of the signal from incandescnet particle
The number required time determines the concentration of the incandescnet particle in sample solution.
In above-mentioned device of the invention, the dense of incandescnet particle can also be determined according to the detection speed of incandescnet particle
Degree, the detection speed is the time required for pre-determined number is reached according to the number of the signal from incandescnet particle and true
Fixed.In addition, above-mentioned device of the invention be configured to reach in the number of the signal from incandescnet particle it is pre-determined
Number untill in a period of, repeat the optical system using photo detection area move portion by each predetermined time interval
Photo detection area position movement, using optical detection part is to the detection of the light from photo detection area and utilizes signal
The detection to the signal from incandescnet particle of processing unit.Predetermined time interval can be any fixed time interval, or
Person can be arranged on the signal from incandescnet particle number reach untill pre-determined number in a period of according to at that time
Untill the detection number of incandescnet particle correct the unit of predetermined time interval, during improving predetermined time interval and measurement
Between optimization.Also, in above-mentioned device of the invention, the number that can be arranged on the signal from incandescnet particle reaches
Estimated to come from incandescnet particle according to the detection number of the incandescnet particle untill at that time in a period of untill pre-determined number
The number of signal reach the unit of the time required for pre-determined number, experimenter can know the detection of incandescnet particle
The time of end.Especially, therefore, in the apparatus of the present, measurement end time display part and/or photogenic granules can be set
Son detection number display part, measurement end time display part display is according to after the detection of the signal from incandescnet particle starts
Estimated as the number from the signal of incandescnet particle detected by signal processing part, signal from incandescnet particle
Number reaches the time untill pre-determined number, and incandescnet particle detection number display part is shown in the letter from incandescnet particle
Number detection start after as the signal from incandescnet particle detected by signal processing part number, according to the structure,
Experimenter can be expected that the time untill the detection of incandescnet particle terminates, therefore be favourable.In addition, in the above-mentioned of the present invention
It is preferably also in device with defined speed or the speed faster than the diffusion translational speed of incandescnet particle carries out utilizing light detection zone
The movement of the position of the photo detection area of domain move portion, can arbitrarily set the translational speed of the position of photo detection area.
The light analysis technology of the present invention is typically used in analysis or parsing protein, peptide, nucleic acid, lipid, sugar chain, amino acid
Or the use of the state of the biological object thing of the particle shape such as organism molecule, virus, the cell such as their condensate in the solution
On the way, but can be used for analyzing or parse abiology particle (such as atom, molecule, micella (micelles), metallic colloid
Deng) state in the solution, it should be recognized that such case falls within the scope of the present invention.
The effect of invention
Sum it up, according to the present invention, using its photo detection area in sample in confocal microscope or multiphoton microscope
, can be according to sample solution in the scanning numerator counts method for the presence for being scanned individually to detect incandescnet particle in this solution
In the concentration of incandescnet particle optimize time of measuring.It should be reached especially by the number of the signal of spontaneous light particle in the future
To pre-determined number be correspondingly set as reaching the number of the precision required by its result with arbitrary experiment or metering,
Expect in the case where the concentration of incandescnet particle is unknown, not based on actual concentration, can also obtain high-precision testing result.
In addition, according to the feature, the situation with carrying out the detection of light and the detection of incandescnet particle in the time of measuring that certain is fixed
Compare, can expect to obtain high-precision result by less repetition test, and can expect needed for being tested or being measured
The reduction of man-hour, labour, time and/or the expense wanted.In addition, in the present invention, due to individually detecting incandescnet particle and true
Its fixed concentration, even therefore concentration is relatively low in sample solution and its light was buried in from other in former method
Incandescnet particle light incandescnet particle, be also capable of detecting when, can observe its presence.It is desirable to the feature being applied to instead
Should rate than the product, the relatively small number of intermediate product of quantity of relatively low reaction detection.
The other purposes and advantage of the present invention will become clear by the explanation of following the preferred embodiment of the present invention
Chu.
Brief description of the drawings
Fig. 1 (A) is the in-built schematic diagram for the light analytical equipment for realizing the light analysis technology of the present invention.Fig. 1's
(B) it is schematic diagram that confocal district's groups knit (viewing area of confocal microscope).Fig. 1 (C) is that the direction for changing speculum 7 makes light
The schematic diagram for the mechanism that the position of detection zone is moved in sample solution.
Fig. 2 (A), (B) is the scanning numerator counts method for illustrating to constitute a part for the light analysis technology of the present invention respectively
In light detection principle schematic diagram and measured luminous intensity time change schematic diagram.
Fig. 3 is the figure for illustrating the principle of the present invention, shows and is obtained for certain sample solution by scanning numerator counts method
Light intensity data (time change of luminous intensity) in temporal sequence.(A) schematically show luminous in sample solution
Light intensity data in the case that the concentration of particle is low, (B) shows a case that the concentration of the incandescnet particle in sample solution is high
Under light intensity data.Fig. 3 (C) is the figure by region for schematically showing photo detection area CV.
Fig. 4 be represent in a flowchart the present invention light analysis technology in processing procedure an embodiment
Figure.
Fig. 5 is to represent that what is performed in the step 30 in Fig. 4 or Fig. 8 flow chart divides according to scanning in a flowchart
Sub-count method presses the figure of the example for the processing procedure that each parsing time interval t is performed.
Fig. 6 (A), (B) is to represent to carry out Brownian movement in incandescnet particle while when crossing photo detection area respectively
And due to moving the position of the photo detection area in sample solution with the fast speed of the diffusion translational speed than incandescnet particle
The illustraton of model of the motion mode of particle when dynamic and incandescnet particle crosses photo detection area.Fig. 6 (C) is that explanation is used for according to sweeping
Retouch the presence that numerator counts method detects incandescnet particle according to measured sequential light intensity data (time change of photon counting)
Fig. 5 described in process step in detection signal signal processing example figure.
Fig. 7 shows the actual measurement example (histogram table) of measured photon counter data, data is smoothed
Obtained from curve (dotted line) and the Gaussian function (solid line) that is fitted in pulse domain of the existence.In figure, add " to make an uproar
The signal of sound " is ignored as by the signal that noise or foreign matter are caused.
Fig. 8 (A) be represent in a flowchart the present invention light analysis technology in processing procedure another reality
Apply the figure of mode.Fig. 8 (B) is to represent the step 20 in Fig. 8 (A) in a flowchart ' between the middle parsing time performed
Every t setting and correcting process example figure.
Fig. 9 (A) shows the fluorchrome for being directed to according to scanning numerator counts method and including 100fM, 1pM, 10pM respectively
Deviation (the CV of the number of detected incandescnet particle in five tentative experiments that ATTO633 each sample solution is performed
Value) time change.Fig. 9 (B) show it is in Fig. 9 (A) five tentative experiments for each sample solution, by each
The detection number of incandescnet particle detected required for time of measuring deviation (the CV values of detection of particles time) relative to luminous
The change of the detection number of particle.
Figure 10 shows that the concentration relative to the incandescnet particle (fluorchrome ATTO633) in sample solution passes through scanning point
The experimental example of the change of the detection speed for the incandescnet particle that sub-count method is detected.
Figure 11 is resulting photon counting (light intensity in the light analysis technology of the fluctuation of conventional calculating fluorescence intensity
Degree) time change example, (A) be the particle in sample concentration be can provide enough measurement accuracy degree feelings
Condition, (B) is the concentration of the particle in sample compared to the situation that the situation of (A) is greatly reduced.
Description of reference numerals
1：Light analytical equipment (confocal microscope)；2：Light source；3：Single-mode fiber (single-mode optical
fiber)；4：Collimation lens；5：Dichronic mirror；6、7、11：Speculum；8：Object lens；9：Microplate；10：Ware (sample solution container)；
12：Condenser (condenser lens)；13：Pin hole；14：Shielding filter；14a：Detection light dichronic mirror；15：Multimode light
Fine (multi-mode optical fiber)；16：Photodetector；17：Mirror deflector；17a：Platform position change device；
18：Computer.
Embodiment
Below, the preferred embodiment of the present invention is described in detail.
The structure of light analytical equipment
The light analysis technology of the present invention can be realized by following light analytical equipment：In basic structure, such as Fig. 1
(A) schematically illustrate it is such, combination be able to carry out the optical system and photodetector of FCS, FIDA etc. confocal microscope and
Into.(A) of reference picture 1, light analytical equipment 1 is by optical system 2~17 and for the action for each several part for controlling optical system
And the computer 18 for obtaining and parsing data is constituted.The optical system of light analytical equipment 1 can be with common confocal microscope
Optical system it is identical, here, exit end of the laser (Ex) for making to launch from light source 2 and propagating in the single-mode fiber 3 in optical fiber
As being launched with the light by the intrinsic NA angular disperseds determined, directional light is turned into by collimater 4, by dichronic mirror 5, instead
The reflection of mirror 6,7 is penetrated, object lens 8 are incided.Microplate 9 typically is configured with the top of object lens 8, the microplate 9, which is arranged with, is filled with 1
The sample container or ware 10 of the μ L of μ L~tens sample solution, sample of the laser projected from object lens 8 in sample container or ware 10
Focus is formed in this solution, the strong region (excitation area) of luminous intensity is formed.Dispersed or dissolved in sample solution as sight
Survey the incandescnet particle of object, be typically attached with the molecules of the luminous marks such as fluorchrome, when incandescnet particle enter it is sharp
When encouraging region, incandescnet particle is energized and discharges light extraction therebetween.The light (Em) discharged is anti-by object lens 8, dichronic mirror 5
Mirror 11 is penetrated to reflect and in the optically focused of condenser 12, pass through pin hole 13.In addition, as understood by the skilled person, pin hole
13 configurations are in the position with the focal position of object lens 8 conjugation, thus only from Jiao of such as Fig. 1 (B) laser schematically shown
The light sent in point region, i.e. excitation area is by pin hole 13, and the light beyond focus face is interrupted.Fig. 1 (B) is illustrated
The focus area of laser be typically light detection zone in this light analytical equipment of the effective volume with 1fL~10fL or so
Domain, is referred to as confocal district's groups and knits.In confocal district's groups are knitted, typically Gaussian shaped profile of the luminous intensity using regional center as summit
Or the distribution of Lorentz lorentz's type, its effective volume is using luminous intensity as 1/e2Face for border generally oblong spheroid volume.So,
Passed through the light of pin hole 13 after dichronic mirror 14a through shielding filter 14 (here, only the light of selection specific wavelength bands into
Point.), multimode fibre 15 is directed to, corresponding photodetector 16 is reached, after electric signal in temporal sequence is converted into
Computer 18 is input to, the processing analyzed for light is carried out in the way of being discussed below.As photodetector 16, preferably make
With the photodetector for the hypersensitivity that can be used in photon counting, thereby, it is possible to detect from incandescnet particle
Light, the faint light for example from one or more fluorchrome molecules.
In addition, in the optical system of above-mentioned light analytical equipment, being additionally provided with the light path for changing optical system and coming
By in photo detection area scanned samples solution, even if the position of focus area (that is, photo detection area) moves in sample solution
Dynamic mechanism.As described for making the mechanism that the position of photo detection area is moved, such as Fig. 1 (C) is schematically illustrated
As, the mirror deflector 17 of the direction of change speculum 7 can be used.The mirror deflector 17 can be with leading to
The galvanomirror device equipped in normal laser scanning type microscope is identical.In addition, in order to realize desired photo detection area
The mobile pattern of position, driving speculum is inclined in phase for the light detection carried out under control of computer 18 with photodetector 16
Turn device 17.The position of photo detection area can be arbitrarily selected from circle, ellipse, rectangle, straight line, curve or combinations thereof
The motion track put (can be set in the program in computer 18 that various mobile patterns can be selected.).In addition, although do not have
Diagram, but it is also possible to make the position of photo detection area move in the vertical direction by moving up and down object lens 8.As above institute
State, according to not being mobile sample solution but the light path of change optical system makes the structure that the position of photo detection area is moved,
Mechanical oscillation, hydrokinetic effect will not be substantially produced in sample solution, mechanical function can be excluded to object of observation
The influence of thing, realizes stable measurement.
In addition, as additional structure, can be provided for moving the level side of microplate 9 microscopical (not shown)
To position with the platform position change device 17a of the ware 10 observed by changing.Can be by the console position change device of computer 18
17a action.
In the case where incandescnet particle is lighted by Multiphoton Absorbtion, it is micro- that above-mentioned optical system is used as multi-photon
Mirror.In this case, light only is discharged in the focus area (photo detection area) of excitation light, therefore pin hole 13 can be removed.
In the case that incandescnet particle is lighted by phosphorescence or scattering, the optical system of above-mentioned confocal microscope can be directly used.
In addition, in the case where the obstructed overdriving light of incandescnet particle is lighted by chemiluminescence, biological luminescence, it is convenient to omit use
In the optical system 2~5 of generation excitation light.And it is possible to set multiple excitation light sources as illustrated in light analytical equipment 1
2, it can be set to that the wavelength of excitation light can be properly selected according to the wavelength of the light of excitation incandescnet particle.Similarly, also may be used
To possess multiple photodetectors 16, in the case of the different a variety of incandescnet particles of wavelength are included in the sample, energy can be set to
It is enough to detect the light from them respectively according to wavelength.
The principle of the present invention
As described in " content of the invention " one column, if be clearly described, the method for the present invention is as follows,
That is, " scanning numerator counts method " is one by one detected come self-luminous by confocal microscope as described above (or multiphoton microscope)
The light of particle, in " the scanning numerator counts method ", repeats the movement of photo detection area, the detection of light and incandescnet particle
Detection untill the number of the signal from incandescnet particle reaches pre-determined number, according to the letter from incandescnet particle
Number number the time required for pre-determined number is reached to determine the concentration of the incandescnet particle in sample solution.Below,
Principle for scanning numerator counts method and the determination incandescnet particle concentration of the present invention is illustrated.
1. scan the principle of numerator counts method
The spectral analysis techniques such as FCS, FIDA the advantage is that required compared with existing biochemical analytical technology
Sample size it is few and can promptly perform inspection.However, in the spectral analysis techniques such as FCS, FIDA, in principle, root
Concentration, the characteristic of incandescnet particle are estimated according to the fluctuation of fluorescence intensity, therefore in order to obtain high-precision measurement result, it is desirable to such as
The concentration or number density of incandescnet particle in sample solution as Figure 11 (A) schematically describes are in measurement fluorescence intensity
During in photo detection area CV all the time exist one or so incandescnet particle level, as shown in the right side of the figure
Significant luminous intensity (photon counting) is consistently detected in Dose times.If the concentration or number density of incandescnet particle are less than
This level, then be simply to enter once in a while in photo detection area CV in incandescnet particle as such as Figure 11 (B) describes
In the case of level, as illustrated in the right side of the figure, only there is significant luminous intensity in a part for time of measuring
Signal (photon counting), it is difficult to accurately estimate the fluctuation of luminous intensity.In addition, with the measurements in photo detection area
All the time in the case that the level in the presence of the incandescnet particle of one or so is greatly reduced compared to the concentration of incandescnet particle, in luminous intensity
In the computing of fluctuation, easily influenceed by background, in order to obtain the significant light intensity data of the amount of progress computing enough
And time of measuring is elongated.
Therefore, present applicant is in Japanese Patent Application 2010-044714 and PCT/JP2011/53481, it is proposed that base
In " the scanning numerator counts method " of new principle, even if the concentration of incandescnet particle is divided less than as described above in spectrum such as FCS, FIDA
In the case of the level required in analysis technology, the characteristics such as the number density or concentration of incandescnet particle can be also detected.
As the processing performed in scanning numerator counts method, if be explicitly described, drive for making light detection zone
The mechanism (mirror deflector 17) of the position movement in domain changes light path, as Fig. 2 is schematically described, while making light
Moved in sample solution, i.e. while by photo detection area CV scanned samples solution, while holding detection zone CV position
The measurement of the detection of row light, i.e. luminous intensity.So, such as Fig. 2 (A) like that, (is in figure during photo detection area CV is moved
Time t0~t2), at the region by one incandescnet particle of presence (t1), light extraction is discharged from incandescnet particle, as in Fig. 2
(B) as describing in, occurs the pulse type of significant luminous intensity (Em) on light intensity data in temporal sequence
Signal.So, the movement and light detection of above-mentioned photo detection area CV position are performed, one by one detect occur therebetween such as figure
The signal (significant luminous intensity) of pulse type illustrated in 2 (B), thus indivedual detection incandescnet particles, and entering to its number
Row is counted, so as to obtain the number of the incandescnet particle existed in measured region or have with concentration or number density
The information of pass.In the principle of described scanning numerator counts method, without being united the calculating such as the fluctuation of fluorescence intensity
The calculation process of meter property, but incandescnet particle is one by one detected, therefore can not as little as lead to even in the concentration of the particle to be observed
In the sample solution for crossing the degree that FCS, FIDA etc. are analyzed with enough precision, the concentration or number with particle can be also obtained
The relevant information of density.
2. the principle of the determination incandescnet particle concentration of the present invention
In scanning numerator counts method as described above, due to being randomly dispersed in sample as the incandescnet particle of object of observation
In this solution, therefore there is deviation (execution every time in the detection number of resulting incandescnet particle in the light detection of certain time of measuring
Detect that number is all different during measurement.).Thus, in order to determine that the detection number of incandescnet particle is removed with precision can allow for or satisfaction
Concentration with the incandescnet particle obtained by the volume by region of the photo detection area in time of measuring etc. utilizes incandescnet particle
Detection number derived from arbitrary characteristic, it is necessary to required for detecting to reach the incandescnet particle of the number required by the precision
Time in perform luminous intensity measurement.On the point, the concentration of the incandescnet particle in sample solution is lower, in certain time of measuring
In the obtained detection number of incandescnet particle it is certainly fewer, therefore deviation becomes big, in order to reach precision can allow for or satisfaction,
Need longer time of measuring.Referring for example to Fig. 3, by scanning numerator counts method for incandescnet particle in certain time of measuring α
In the case of the relatively low solution of concentration ratio (A) and the higher solution of incandescnet particle concentration (B) detection incandescnet particle, such as by Fig. 3
(A), (B) be compared as understanding, as illustrated in (B) such as Fig. 3 for incandescnet particle concentration it is higher
The obtained light intensity data in temporal sequence of solution in detect (A) than Fig. 3 it is illustrated as incandescnet particle it is dense
Spend the signal of the incandescnet particle more than the situation than relatively low solution.Thus, for example, can allow for determining for reaching for acquisition
The time of measuring of the detection number of incandescnet particle required by the precision of the characteristics such as concentration, when the solution of (A) for Fig. 3 needs
Between in the case of α, for the solution of Fig. 3 (B), time β is just enough.Further it is assumed that the solution at (A) for Fig. 3 only exists
In the case of performing measurement in time β, the deviation of the detection number of incandescnet particle becomes big, and the error of the result of concentration etc. is possible to big
To unallowable degree.
However, when the incandescnet particle concentration in sample solution is unknown, luminous intensity is carried out in the time of measuring of certain fixation
Measurement in the case of, time of measuring is set to long enough in order to guard against the concentration of incandescnet particle low.In this case,
When the concentration of incandescnet particle in sample solution is high, become exceed the spies such as concentration are determined with precision can allow for or satisfaction
Time test constantly luminous intensity required for property.In addition, the incandescnet particle concentration in sample solution is assumed less than experimenter
Concentration and set time of measuring it is not enough in the case of, cause the error of result to become big.
Therefore, in the present invention, it is not according to measurement (that is, the light that luminous intensity is performed in the time of measuring of certain fixation
Detection) obtained by light intensity data detection photogenic granules subnumber, but repeat to make photo detection area move and carry out light intensity
The measurement of degree and the detection of incandescnet particle signal reach that pre-determined number is until the number of the signal from incandescnet particle
Only, the number of signal of the metering from incandescnet particle reaches the time required for pre-determined number, is come from according to described
The number of the signal of incandescnet particle reaches the time required for pre-determined number to determine the concentration of incandescnet particle.According to institute
Structure is stated, in the case that the incandescnet particle concentration in sample solution is high, the time required for luminous intensity measurement can be shortened,
In the case that incandescnet particle concentration in sample solution is low, can test constantly luminous intensity (that is, lighted until result must be reached
Particle concentration) required by precision photogenic granules subnumber untill.Moreover, should by the number of the signal of spontaneous light particle in the future
The pre-determined number reached is previously set to reach the photogenic granules subnumber of the precision required by result, is up to result and is wanted
The photogenic granules subnumber for the precision asked is reflected as required for the number of the signal from incandescnet particle reaches pre-determined number
Time, therefore, it is possible to expect that the concentration value of the incandescnet particle determined according to the time has precision can allow for or satisfaction.
Specifically, as following by according to present invention determine that incandescnet particle concentration value with from incandescnet particle
The number of signal reaches the association in time required for pre-determined number.That is, certain incandescnet particle concentration C sample
In solution, in the case of photo detection area is made in time τ with sweep speed u movements, set when by the sectional area of photo detection area
During for S ((C) of reference picture 3), the number X of the signal of detected light is
X=CSu τ NA…(1)。
Here, NAIt is Avogadro's number.Thus, when the number for setting the signal from incandescnet particle reach it is pre-determined
When number XE needs time T, the concentration C of incandescnet particle is provided as time T function by following formula.
C=XE/ (STuNA)…(2)
In addition, in formula (2), required for pre-determined number XE is reached according to the number of the signal from incandescnet particle
Time T and incandescnet particle detection number XE, by following formula provide time per unit particle detection speed V,
V=XE/T ... (3)
Therefore the concentration C of incandescnet particle is expressed as
C=V/ (SuNA)…(4)。
In the formula (4), the concentration C and detection speed V of incandescnet particle are into single order ratio, due to easily knowing incandescnet particle
Concentration C and detection speed V corresponding relation, therefore in actual experiment, it is possible to use detection speed V determines incandescnet particle
Concentration C.(with reference to following embodiments)
Handle operating process
In the embodiment that the light of the invention using the light analytical equipment 1 illustrated in Fig. 1 (A) is analyzed, specifically
Say, perform following process, the modulation of the sample solution of (1) comprising incandescnet particle, the measurement of the luminous intensity of (2) sample solution and
The detection of incandescnet particle, counting processing, and (3) concentration such as calculate at point analysis.
(1) modulation of sample solution
In the light analysis technology of the present invention, as long as molecule dissolved as the particle of object of observation etc. is in sample solution
In disperse and the particle of random motion in the solution, then can be arbitrary, for example can be protein, peptide, nucleic acid, lipid,
Organism molecule, virus, cell or the metallic colloids such as sugar chain, amino acid or their condensate, other abiology particles etc.
(sample solution is typically the aqueous solution, but is not limited to other arbitrary liquid of this or organic solvent etc.).
In addition, can be its own luminous particle as the particle of object of observation, or or it addition of hair in any way
Cursor knows the particle of (fluorescence molecule, phosphorescent molecules, chemistry, bioluminescent molecules).
, can be with due to can determine the incandescnet particle concentration in sample solution, therefore for example in addition, in the present embodiment
The concentration of incandescnet particle by the solution comprising the unknown composition of concentration, before and after dissociation reaction, intermolecular interaction is combined
Solution of change etc. be used as sample solution, carry out solution in composition concentration determination, detection reaction, interaction have
The degree of nothing, progress.
(2) measurement of the luminous intensity of sample solution and the detection of incandescnet particle, counting
Fig. 4 is this embodiment party for representing to perform using the light analytical equipment 1 illustrated in Fig. 1 (A) in a flowchart
The measurement of the luminous intensity of sample solution in formula and the detection of incandescnet particle, an example for counting processing.In the example of the figure
In son, if be explicitly described, photo detection area is repeated by each parsing time interval t (predetermined time interval)
The movement of position, the detection of light from photo detection area, the detection of signal from incandescnet particle and detected hair
This series of processes of counting of the signal of light particle reaches that end population XE is (luminous until detected photogenic granules subnumber X
The population pre-determined number to be reached) untill.Further, it will be understood that to be realized by the processing of computer 18 action
A series of processing described below and structure.[the survey of analysis, the luminous intensity of (4) sample solution in (3) concentration calculating etc.
It is identical in the amendment example that detection, the counting of amount and incandescnet particle are handled.]
(i) initial setting
In operation processing, specifically, first in the injecting sample solution of ware 10 to microplate 9 and it is positioned in microscopical
After on platform, instruction is started to what the measurement of the input light intensity of computer 18 and detection, the counting of incandescnet particle were handled in user
When, as initial setting, computer 18 terminate population XE setting (step 10) and parsing time interval t setting
(step 20).Terminating population XE and parsing time interval t can arbitrarily be set by user.It can refer to and use photogenic granules
Solution known to the concentration of son can reach the preliminary experiment institute of the precision required by the end value of the concentration of incandescnet particle
Obtained result come suitably determine terminate population XE (with reference to embodiment described later).It is used as parsing time interval t, Ke Yikao
Consider aftermentioned Fig. 5 processing speed in device 1 etc. suitably to set with reaching end to photogenic granules subnumber (X) since processing
The time of population (XE) is compared to fully short arbitrary time interval.In addition, terminating population XE and parsing time interval t
Can will come pre- by reference to the result obtained by solution progress preliminary experiment known to the concentration using incandescnet particle respectively
The value storage first determined in the device 1, and automatically or by the selection of user uses stored described value.
(ii) detection of photogenic granules subnumber
So, when finish bundle of particle number XE and parsing time interval t setting, in incandescnet particle as following
Total number X (tn) reach before end population XE (step 50), repeat following steps by each parsing time interval t：
In parsing time interval t using the measurement processing for the luminous intensity for scanning the progress of numerator counts method, based on measured light intensity
The detection of the incandescnet particle signal of degrees of data and photogenic granules subnumber x detection (step 30)；And to detecting in step 30
The photogenic granules subnumber x gone out is accumulated and estimated the processing (step 40) of the total number X (tn) of incandescnet particle.In addition, repeating to hold
Before the processing of row step 30~50, a series of Ts (step 25) between can storing at the beginning of processing.Below, step is described in detail
Rapid 30~50 processing.
(a) measurement of luminous intensity
Fig. 5 is the example for representing the processing procedure in step 30 in a flowchart.With reference to the figure, in the place of step 30
During reason, first while driving mirror deflector 17 carries out movement (sample of the position of photo detection area in sample solution
Scanning in this solution), while carrying out the measurement (Fig. 5 step 100) of luminous intensity in parsing time interval t.At the place
In reason, typically according to the program being stored in storage device (not shown) (in order that the position of photo detection area is in sample
Moved in solution and change the process of light path, irradiated to photo detection area and encourage the process (only when needed) of light and examined in light
The process of the light from photo detection area is detected in the movement for the position for surveying region), proceed by the light detection in sample solution
The irradiation (only when needed) and the measurement of luminous intensity of excitation light at region.When start measurement when, first computer 18 according to
Under the control of the processing action carried out according to program, from the light of the excitation wavelength of the incandescnet particle in the injection sample solution of light source 2, and
And mirror deflector 17 drives speculum 7 (galvanomirror) to perform movement of the position of photo detection area in ware 10, with this
Meanwhile, photodetector 16, which is converted to the light being sequentially received, is sent to computer 18 after electric signal, computer 18 is according to any
Mode according to the signal generation sent light intensity data in temporal sequence and preservation.Typically photodetector 16 is energy
The hypersensitivity photodetector of the arrival of a photon is enough detected, therefore, the detection of light is with every the defined unit interval
(BIN TIME), the photon counting for example performed every the mode of the 10 μ s numbers for measuring the photon for coming photodetector successively,
The data of luminous intensity in temporal sequence can be photon counter data in temporal sequence.
On the translational speed of the position of photo detection area, in scanning numerator counts method, in order to quantitatively accurately
Light intensity data in temporal sequence according to measuring detects individually incandescnet particle, preferably by the measurement process of luminous intensity
In the translational speed of position of photo detection area be set as shifting caused by random motion, the i.e. Brownian movement than incandescnet particle
Move fireballing value.Translational speed in the position of the photo detection area feelings slower than the movement carried out by Brownian movement of particle
Under condition, as Fig. 6 (A) schematically describes, particle is randomly moved in region, thus, and luminous intensity randomly becomes
(the excitation luminous intensity of photo detection area is reduced outward using the center in region as summit for change.), it is difficult to it is determined that with each photogenic granules
The change (signal for representing the light from incandescnet particle) of the corresponding significant luminous intensity of son.It is therefore preferable that such as Fig. 6
(B) as describing, particle crosses photo detection area CV generally in straight line, thus, in light intensity data in temporal sequence
In, the curve of the change of luminous intensity corresponding with each particle is the excitation light intensity with Fig. 6 (C) topmost as illustrated
Degree distribution is roughly the same substantially to hang mitriform, and the translational speed of the position of photo detection area is set to than particle because of Brownian movement
And the average translational speed (diffusion translational speed) carried out is fast, enabling be readily determined each incandescnet particle and luminous intensity it
Between correspondence.
Specifically, the incandescnet particle with diffusion coefficient D due to Brownian movement by radius r photo detection area
Required time Δ τ is according to the relational expression of following Mean square displacement when (confocal district's groups are knitted),
(2r)2=6D Δs τ ... (5)
Turn into
Δ fourth=(2r)2/ 6D ... (6),
Therefore, Vdif is substantially for the speed (diffusion translational speed) of incandescnet particle movement because of Brownian movement
Vdif=2r/ Δs τ=3D/r ... (7).
Therefore, the translational speed of the position of photo detection area be referred to the Vdif be set as in contrast fully it is fast
Value.It is D=2.0 × 10 for example in the diffusion coefficient of anticipation incandescnet particle-10m2In the case of/s or so, r is being set as 0.62 μm of left side
When right, Vdif is 1.0 × 10-3M/s, therefore, it can the translational speed of the position of photo detection area being set as its more than 10 times
Such as 15mm/s.In addition, in the case where the diffusion coefficient of incandescnet particle is unknown, the position of various photo detection areas can be set
The translational speed put, performs the curve of the change for finding luminous intensity and (is typically and excitation light for the curve of anticipation repeatedly
Intensity distribution is roughly the same) condition preliminary experiment, come the translational speed of the position that determines suitable photo detection area.
(b) detection of signal corresponding with incandescnet particle
When by above-mentioned processing parsing time interval t obtain incandescnet particle in sample solution in temporal sequence
During light intensity data, handled by computer 18 according to the program being stored in storage device, to perform the light intensity number of degrees
According to the detection of upper signal corresponding with light from incandescnet particle.
In light intensity data in temporal sequence, track such as Fig. 6 when an incandescnet particle is by photo detection area
(B) shown in be substantially linear like that in the case of, the change of the luminous intensity of signal corresponding with the particle have reflection (by
What optical system was determined) ((C) of reference picture 6 is most upper for the curves for substantially hanging mitriform of light intensity distributions in photo detection area
Portion).Thus, scanning numerator counts method in, substantially can more than the luminous intensity of the threshold value suitably set it is lasting when
Between width when being in defined scope, be determined as that the signal and a particle of the curve with the luminous intensity pass through light detection zone
The situation correspondence in domain, carries out the detection of an incandescnet particle.Moreover, the luminous intensity duration width more than threshold value does not exist
Signal in defined scope is judged as the signal of noise or foreign matter.In addition, the luminous intensity of photo detection area can divided
When cloth is assumed to Gaussian Profile, i.e.,
I=Aexp (- 2t2/a2) ... (8),
Count making formula (8) be fitted the curve curve of background (can clearly be judged as be not) of significant luminous intensity
When the intensity A and width a calculated is in defined scope, the curve of the luminous intensity can be judged to passing through with a particle
The situation correspondence of photo detection area, carries out the detection of an incandescnet particle.(intensity A and width a are in the letter outside defined scope
Number it is judged as ignoring in the signal of noise or foreign matter, analysis that can be behind etc..)
It is used as an example of the more specifically method of the processing for the detection that incandescnet particle is carried out according to sequential light intensity data
Son, carries out smooth (smoothing) place to sequential light intensity data (Fig. 6 (C), topmost " testing result (untreated) ") first
Manage (Fig. 5 step 110, the middle and upper part " smooth " of Fig. 6 (C)).What what incandescnet particle was sent discharged just probabilityly,
There may be the missing of data value in the small time, therefore by the smoothing processing, it can ignore that data as described above
The missing of value.It can be for example smoothed by the method for moving average.In addition it is also possible to according to obtain light intensity data when
The translational speed (sweep speed) of the position of photo detection area, BIN TIME, suitably setting perform smoothing processing when parameter,
Such as average data amount check, the number of times of rolling average are carried out once in the method for moving average.
Then, in the sequential light intensity data after smoothing processing, in order to detect that the signal of significant pulse type is (following
Time zone (pulse domain of the existence) present in referred to as " pulse signal "), to the sequential light intensity data after smoothing processing
Time Calculation once differentiation value (step 120).Middle and lower part " the time of the time diffusion value of sequential light intensity data such as Fig. 6 (C)
It is big in the change of the change moment value of signal value as differential " is illustrated, therefore by referring to the time diffusion value, energy
Enough beginning and ends for advantageously determining significant signal.
Then, on sequential light intensity data, significant pulse signal is detected successively, whether is the signal that judgement is detected
It is signal corresponding with incandescnet particle.Specifically, first in the time diffusion value in temporal sequence of sequential light intensity data
In data, reference time differential value is searched for and determines the beginning and end of a pulse signal successively, determines pulse domain of the existence
(step 130).When a pulse domain of the existence is determined, for the sequential luminous intensity after smooth in the pulse domain of the existence
Data hang the fitting (" the hanging bell Function Fitting " of the bottom of Fig. 6 (C)) of bell function, calculate the arteries and veins for hanging bell function
(least square method) phase when the intensity I peak of the peak value (maximum) of punching, pulse width (full width at half maximum (FWHM)) Wpeak, fitting
The parameter such as relation number (step 140).In addition, the bell function that hangs of fitting is typically the Gaussian function as formula (8), but
It can be Lorentz lorentz's type function.Moreover, judging whether the parameter for hanging bell function calculated is in for an incandescnet particle
In the scope that what the pulse signal detected during by photo detection area was described hang contemplated by the parameter of the curve of bell, i.e. arteries and veins
Whether peak strength, pulse width, the coefficient correlation of punching are respectively in prescribed limit etc. (step 150).So, such as Fig. 7
Shown in the left side like that, the parameter for hanging bell function for being judged as calculating is in for letter corresponding with an incandescnet particle
Thus signal determining in the range of number contemplating, detects an incandescnet particle to be signal corresponding with an incandescnet particle,
So as to be counted as an incandescnet particle (by population x cumulative 1.Step 160).On the other hand, as shown in Fig. 7 the right that
Sample, pulse signal of the parameter for hanging bell function calculated not in the range of imagination is ignored as noise.
Above-mentioned steps 130 can be performed repeatedly in the whole region of the sequential light intensity data in parsing time interval t
Search, judgement and the counting (step 170) of pulse signal in~160 processing.In addition, according to sequential light intensity data
Do not detect that the processing of the signal of incandescnet particle is not limited to above-mentioned process, can be performed by arbitrary method.When in parsing
At the end of all sequential light intensity data middle arteries in time interval t rush the search of signal, step 30 terminates, and performs step 40.
(c) calculating of the detection sum of incandescnet particle
So, when detecting to parse the photogenic granules subnumber x in the sequential light intensity data in time interval t, under
Formula calculates the detection total number X (tn) (Fig. 4 step 40) of incandescnet particle.
X(tn)=X (tn-1)+x…(9)
In addition, X (tn-1) be the particle detected untill previous parsing time interval t detection sum, its is initial
Value is 0.Then, step 30~40 are repeated until the detection total number X (tn) of incandescnet particle reaches by each parsing time interval t
Untill population XE is terminated, i.e., untill following formula is set up (step 50).
X(tn)≥XE…(10)
So, during step 30~50 are repeated, when formula (10) is set up, the luminous intensity of sample solution is terminated
Measurement and incandescnet particle detection, count processing.At the end of the processing repeatedly of step 30~50, it can store and terminate
Time TE (step 60).
(d) display of photogenic granules subnumber and measurement end time
(it can be set up until formula (10) during step 30~50 are performed repeatedly by each parsing time interval t and be
Only), at the end of detection total number X (tn) and/or measurement that incandescnet particle is shown on the first-class display of monitor of computer 18
Between TE or measurement remaining time Tr.According to the structure, it can predict what is be carrying out by observing these displays in user
What time measurement terminates to be favourable on this point.
In the case where performing display as described above, in the invalid situation of the judgement Chinese style (10) of Fig. 4 step 50
Under, perform each processing shown in dotted line in figure.Specifically, first by the newest incandescnet particle estimated in step 40
Detect total number X (tn) display over the display (step 52).In addition, in the case where performing step 30~50 repeatedly,
Update the value of the detection total number X (tn) of incandescnet particle untill at that time.Then, end time TE or measurement are measured in order to calculate
Remaining time Tr and the processing of calculation procedure 30~50 start after incandescnet particle detection speed v (step 54).It can pass through
Following formula provides the detection speed v of incandescnet particle till now.
V=X (tnThe Ts of)/(Tp mono-) ... (11)
Here, at the time of Tp is current.So, it is remaining by following formula estimation measurement using the detection speed v of incandescnet particle
Time Tr (time that the processing to step 30~50 terminates),
Tr=(XE-X (tn))/v…(12)
In addition, passing through following formula estimation measurement end time TE (time that the processing of step 30~50 terminates) (step 56).
TE=Tp+Tr ... (13)
Then, estimated measurement end time TE or measurement remaining time Tr are shown into (step over the display
58).In addition, in the case where performing step 30~50 repeatedly, updating the value having shown that.In addition, at X (tn)=0
When, not arithmetic expression (12) or (13) and Tr and TE can be shown as not clear.
In addition, as has been described, repeating above-mentioned Fig. 4 step 30 by each parsing time interval t
~50, the processing of Fig. 5 step 100~170.On the point, step can performed untill the start to finish from measurement
Also the measurement of the luminous intensity of Fig. 5 step 100 is consecutively carried out during signal transacting step beyond 100.That is, in Fig. 4
In~5 processing cycle, when one circulation parsing time interval t in step 100 when being measured of luminous intensity, directly
The measurement of the luminous intensity for the step 100 being consecutively carried out in the parsing time interval t of subsequent cycle, while the root in computer 18
Performed according to the light intensity data that is got in the parsing time interval t of the circulation completed the signal of incandescnet particle detection,
The processing of counting.Thus, the detection of incandescnet particle is reached in real time, is counted.
(3) analysis of concentration calculating etc.
So, when photogenic granules subnumber reaches end population, it is possible to use photogenic granules subnumber reaches that end population is
Time T (=TE-Ts) or the other information obtained according to the signal of detected incandescnet particle only, are calculated to perform concentration
Deng analysis (step 70).
As has been described, using formula (3), according to the time T and end particle reached untill terminating population
Number XE calculates the detection speed V of particle, and according to the detection speed V of particle, the dense of incandescnet particle is determined using the relation of formula (4)
Degree.
Furthermore, it is possible to according to excitation light or the wavelength of detection light, the numerical aperture of lens, the adjustment state of optical system,
The sectional area S by region of photo detection area in theory in estimation formula (1)~(4), but can also be according to by such as
The number for the incandescnet particle that lower method is detected and the concentration of the incandescnet particle of contrast solution are determined, and this method is to pass through
Experiment, such as it is identical with the measurement with the sample solution to be checked for solution known to the concentration of incandescnet particle (contrast solution)
Condition carry out the measurement of above-mentioned illustrated luminous intensity, the detection of incandescnet particle, count.Specifically, for example for luminous
The contrast solution of the concentration C of particle, the photogenic granules in setting with the measurement of the translational speed uo luminous intensities performed in certain time τ o
When the detection number of son is N, the sectional area S by region of photo detection area is given by the following formula.
S=N/ (CNA·uo·τo)…(14)
In addition, as contrast solution, the solution of multiple various concentrations of incandescnet particle can be prepared, held for each solution
Row measurement, and calculated S average value is used as the sectional area S of photo detection area.Furthermore, it is possible to not based on above-mentioned
Method and the sectional area S that photo detection area is provided by arbitrary method, such as by using FCS, FIDA.In addition, at this
, can be by various standards on the Move Mode of contemplated photo detection area in the light analytical equipment of embodiment
The information of relation (formula (14)) between the concentration C of incandescnet particle and the number N of incandescnet particle is prestored to computer 18
In storage device, device user implement light analysis when can suitably with the relation stored information.
(4) measurement of the luminous intensity of sample solution and the detection of incandescnet particle, the fixed case for counting processing
In the measurement of the luminous intensity of above-mentioned sample solution and detection, the counting of incandescnet particle are handled, as other
Mode, parsing time interval t may not be fixed value but it be modified according to the detected status of incandescnet particle.Figure
8 (A) represents to be configured to include repairing parsing time interval t according to the detected status of incandescnet particle in a flowchart
The measurement and detection, the counting processing, Fig. 8 (B) of incandescnet particle of the luminous intensity of the sample solution of positive processing (step 20 ')
In a flowchart represent step 20 ' in parsing time interval t calculation process.In addition, in Fig. 8 (A), pair with figure
The additional identical number of steps of 4 identicals processing.
With reference to the figure, in Fig. 8 processing, whenever all being corrected when being measured for the luminous intensity in parsing time interval t
Parsing time interval t (step 20 ').It is configured in addition, the processing of example illustrated is particularly from starting to photogenic granules subnumber
Reach in the one-shot measurement untill terminating population XE, only with pre-determined times N (hereinafter referred to as " renewal pre-determined number ".)
The processing cycle for perform the measurement of luminous intensity and the detection of incandescnet particle, counting.Specifically, first, as initial setting,
After the setting (step 10) and time started Ts storage (step 25) for terminating population XE, luminous intensity is initially being performed
Measurement and incandescnet particle detection, count processing when, luminous intensity measurement and incandescnet particle detection, counting
When the execution number of times k of processing cycle is 0, the initial value to that can be arbitrarily set to parsing time interval t impartings be (reference picture 8
(B) step 200,210).Then, the execution number of times of processing cycle increases by 1 (step 270), same with the processing described in Fig. 4
The processing (step 30~50) that ground performs the measurement of the luminous intensity in parsing time interval t and the detection of incandescnet particle, counted.
So, as the photogenic granules subnumber x (=X (t for obtaining initial circulation1)) when, detection of particles speed v (step 54) is estimated successively, surveyed
Measure remaining time Tr (step 56).In addition, with Fig. 4 situation likewise it is possible in the first-class display of monitor of computer 18
The detection total number X (tn) and/or measurement end time TE of upper display incandescnet particle or measurement remaining time Tr (step 52,58).
In addition, photogenic granules subnumber is reached when finishing bundle of particle number XE in initial processing cycle, directly terminate the measurement of luminous intensity with
And detection, the processing (step 50) of counting of incandescnet particle.
After initial processing cycle, parsing time interval t amendment and the situation identical with Fig. 4 is repeated
The measurement of luminous intensity and the detection of incandescnet particle, the processing cycle counted (step 20 ', 30~58) are until photogenic granules subnumber reaches
Untill terminating population XE.Now, the parsing time interval t step 20 of amendment is being carried out ' in, first, determining now
Untill the photogenic granules subnumber X (tn) that detects whether be 0 (step 220)., can be by the parsing of previous circulation at X (tn)=0
Time interval t is multiplied by m times (m is more than 1 positive number).At X (tn)>When 0, using measuring remaining time Tr, update pre-determined number N
And the execution number of times k of processing cycle, pass through following formula estimation parsing time interval t (step 240).
T=Tr/ (N-k) ... (15)
Furthermore, it is possible to lower limit is set for the parsing time interval t estimated, can be in the case where parsing time interval t be less than
Parsing time interval t is set as lower limit tmin (step 250,260) during limit value tmin.
As described above, according to the mode being modified to parsing time interval t, as in the sample solution of object of observation
The detected status of incandescnet particle is reflected as measuring remaining time Tr, therefore will be parsed according to the detected status of the incandescnet particle
Time interval t is optimized.
So, according to the above-mentioned present invention, it is scanned to detect individually in using photo detection area to sample solution
In the scanning numerator counts method of incandescnet particle, due to being the detection for the measurement and incandescnet particle for repeating luminous intensity, counting
Such mode untill the number of the signal from incandescnet particle reaches pre-determined number, thus with sample solution
Incandescnet particle concentration correspondingly the measurement of luminous intensity and the detection of incandescnet particle, count required for time (during measurement
Between) increase or decrease.Moreover, the pre-determined number that should be reached by the number of the signal of spontaneous light particle in the future is prior
It is set as that the number of the precision required by result can be reached, can just can allow for without the time of measuring for requiring more than needs
Or satisfaction result.
In order to verify the effectiveness of the invention of described above, tested as follows.Further, it will be understood that for
Under embodiment be used to illustrate effectiveness of the invention, and be not to limit the scope of the present invention.
Embodiment 1
Using the solution for being dissolved with fluorchrome molecule as sample solution, incandescnet particle is carried out using numerator counts method is scanned
Detection and counting, and confirm the feature of the deviation of the number of detected incandescnet particle, and according to the structure of the present invention,
Demonstrate the concentration independent of incandescnet particle, and can the deviation of result be suppressed small and can be during as short as possible
Inside accurately detect the concentration of incandescnet particle.
As sample solution, modulate by ATTO633 (Sigma-Aldrich companies, Cat.No.18620) using concentration as
100pM, 10pM, 1pM, 100fM mode are dissolved in solution resulting in the PBS comprising 0.05%Tween20.
In the measurement of light, as light analytical equipment, the optical system and the list of Photo Counting System for possessing confocal fluorescence microscopy are utilized
Molecular fluorescence measurement apparatus MF-20 (Olympus Co., Ltd), according in above-mentioned " measurement of (2) (ii) (a) luminous intensity "
In illustrated mode, obtain sequential light intensity data (photon counter data) for each above-mentioned sample solution.Now,
Encourage light to use 633nm laser, the photogenerated sequential light intensity of 660nm-710nm wavelength band is measured using bandpass filter
Degrees of data.The photo detection area in sample solution is set to be moved with the translational speed of 30mm/ seconds.In addition, BIN TIME are set
For 10 μ s, five measurements have been carried out for each solution.In addition, this experiment is the feature for confirming the number deviation of incandescnet particle
Experiment, therefore be not the measurement that luminous intensity is performed by each parsing time interval, but be enough to capture incandescnet particle
The measurement of luminous intensity is carried out in the time of the feature of number deviation.(in fact, for 100pM, 10pM, 1pM solution, measurement
Time is set to 2 seconds, for 100fM solution, and time of measuring is set to 20 seconds.)
After the measurement of above-mentioned luminous intensity, according to it is above-mentioned " (2) (ii) (b) signal corresponding with incandescnet particle
Processing procedure described in detection ", smoothing processing is implemented to sequential light intensity data obtained above, in the data after smooth
The beginning and end of pulse signal is determined, each pulse signal of Gaussian function fitting is made by least square method afterwards, it is determined that (Gauss
In function) peak strength, pulse width (full width at half maximum (FWHM)), coefficient correlation.Then, only the pulse for meeting following conditions is believed
Number it is determined as signal corresponding with incandescnet particle,
20μs<Pulse width<400μs
Peak strength>1.0[pc/10μs]…(A)
Coefficient correlation>0.95,
On the other hand, the pulse signal for being unsatisfactory for above-mentioned condition is ignored as noise.
Fig. 9 (A) show scanning numerator counts method in relative to time of measuring length the photogenic granules detected
The change of the deviation of subnumber.Transverse axis in figure is the length of time of measuring, and the longitudinal axis is the photogenic granules detected in being measured at five times
The CV values (=standard deviation/average × 100%) of subnumber, each point represents the CV values in the length of corresponding time of measuring.Such as from this
All it is that the longer CV values of time of measuring are lower for above-mentioned 10pM, 1pM, 100fM sample solution as figure understands.Separately
Outside, the value of three sample solutions is compared it will be apparent that, concentration is lower, CV values is become sufficiently small the spent time and gets over
It is long.Thus, the deviation convergence for showing the concentration difference of the incandescnet particle in sample solution and making incandescnet particle detect number
It is different in the time of measuring in admissible scope.
On the other hand, Fig. 9 (B) shows the acquisition for detecting number relative to incandescnet particle in scanning numerator counts method
The change of the deviation of time of measuring (detection of particles time) required for each detection number.Transverse axis in figure is detected hair
Light bead subnumber, each point represents the time of measuring (detection of particles required for detecting corresponding photogenic granules subnumber in being measured at five times
Time) CV values (=standard deviation/average × 100%).As understood from the figure, on above three sample solution,
When photogenic granules subnumber is more than 30, dependence of the CV values to incandescnet particle concentration of detection of particles time there's almost no.The feelings
Shape implys that carries out the measurement of luminous intensity until the number of the signal from incandescnet particle is changed into energy in scanning numerator counts method
Untill the number for enough reaching precision required by result, pass through the measurement according to required for the incandescnet particle obtained now detects number
Time performs the analysis of concentration etc., can obtain the few result of deviation.
In addition, (B) of reference picture 9 result, is being that 100fM solution and 10pM solution are obtained for pigment molecular concentration
To light intensity data in, by incandescnet particle detection number reach that time of measuring and detection of particles speed untill 130 are compared
Compared with.As a result it is as follows.
[table 1]
10pM ATTO633
[table 2]
100fM ATTO633
As understood according to above-mentioned table, scenario described below is shown：It can permit in order that the deviation of result is converged on
Perhaps degree, in the case of 100fM solution, time of measuring needs more than 14 seconds, on the other hand, in 10pM solution
In the case of, time of measuring 0.18 second or so.The situation represents to suppress small and height by deviation in scanning numerator counts method
The time of measuring required for result is obtained to precision according to the different and different of incandescnet particle concentration.Moreover, showing following feelings
Shape：By the measurement of the execution luminous intensity according to the present invention untill the detection number of incandescnet particle reaches pre-determined number
Mode, in time of measuring corresponding with incandescnet particle concentration carry out luminous intensity measurement, can avoid taking over needs
Time measure.Particularly it should be understood that when in the case that incandescnet particle concentration is high measurement significantly can be shortened
Between.
Also, untill Figure 10 shows to reach 150 according to the detection number of above-mentioned each sample solution for the drafting of each concentration
The figure that the detection of particles speed that time of measuring is calculated is obtained.As from figure understanding, scenario described below is implied that：Particle is examined
Degree of testing the speed is proportional to pigment concentration, and the relation of above-mentioned formula (4) is set up.Thus, it is advance according to detection of particles speed or detection
Time of measuring required for the incandescnet particle of the number of decision, can determine the concentration of the incandescnet particle in sample solution.
So, as from the understanding of the result of above-described embodiment, according to the invention described above, in scanning numerator counts method
In, it is not photo detection area is moved while performing measurement and the photogenic granules of luminous intensity in the time of measuring of certain fixation
The detection of son, but move photo detection area while performing the measurement of luminous intensity and the detection of incandescnet particle until coming
Untill the number of the signal of spontaneous light particle reaches pre-determined number, by such mode, the deviation of result is suppressed
It is small and optimize time of measuring, and can be used in detection reach precision required by result number incandescnet particle survey
Amount terminates in a period of as short as possible.Especially, the present invention individually detects the signal of incandescnet particle, even if therefore sample is molten
The incandescnet particle concentration of liquid can also carry out the detection of incandescnet particle less than the concentration range required by the light analysis technologies such as FCS,
The feelings that the rare or expensive sample that the feature is commonly used in medical science, the research and development field of biology is analyzed
It is favourable under condition., also being capable of the phase in the case that incandescnet particle concentration in sample solution is unknown in addition, by the present invention
Labour, time or expense of the reduction for the repetition test of time of measuring are treated, therefore, it is possible to expand answering for scanning numerator counts method
Use scope.For example, need not be due to when incandescnet particle concentration be measurement that is unknown and setting the length for exceeding needs in sample solution
Between to prevent the extremely low situation of preparation light particle concentration.
Claims (14)
1. the optical system of a kind of use confocal microscope or multiphoton microscope come detect come comfortable sample solution in disperse and
The light of the incandescnet particle of random motion and the method analyzed, it is characterised in that comprise the following steps：
Photo detection area moving step, makes the photo detection area of above-mentioned optical system by changing the light path of above-mentioned optical system
Position moved in above-mentioned sample solution；
Light detecting step, makes the position of above-mentioned photo detection area mobile in above-mentioned sample solution while detecting from above-mentioned
The light of photo detection area；
Sequential light intensity data generation step, according to the detected photogenerated from above-mentioned photo detection area in temporal sequence
Sequential light intensity data；
Smoothing step, is smoothed to above-mentioned sequential light intensity data, until can ignore that above-mentioned incandescnet particle institute
The light sent is within the small time untill the missing of issuable data value；And
On incandescnet particle detecting step, the above-mentioned sequential light intensity data after smoothing processing, by with the song for substantially hanging mitriform
Line, luminous intensity change with time and be detected as the signal of the light from each above-mentioned incandescnet particle one by one, wherein this substantially hangs
Light intensity distributions that the curve of mitriform reflects in above-mentioned photo detection area and with being in for corresponding with an incandescnet particle
Signal contemplate in the range of parameter,
Wherein, above-mentioned photo detection area moving step, above-mentioned smooth detecting step, the generation of above-mentioned sequential light intensity data are repeated
Step, above-mentioned smoothing step and above-mentioned incandescnet particle detecting step are until the signal of the light from above-mentioned incandescnet particle
Untill number reaches pre-determined number, reached according to the number of the signal of the light from above-mentioned incandescnet particle pre-determined
Time required for number, to determine the concentration of the above-mentioned incandescnet particle in above-mentioned sample solution.
2. according to the method described in claim 1, it is characterised in that
In a period of untill the number of the signal of the light from above-mentioned incandescnet particle reaches pre-determined number, by each rule
Fixed time interval repeats above-mentioned photo detection area moving step, above-mentioned smooth detecting step, above-mentioned sequential light intensity data
Generation step, above-mentioned smoothing step and above-mentioned incandescnet particle detecting step.
3. according to the method described in claim 1, it is characterised in that
The concentration of the above-mentioned incandescnet particle in above-mentioned sample solution, the detection are determined according to the detection speed of above-mentioned incandescnet particle
Speed is the time according to required for the number of the signal of the light from above-mentioned incandescnet particle reaches pre-determined number and true
Fixed.
4. according to the method described in claim 1, it is characterised in that further comprising the steps of：
In a period of untill the number of the signal of the light from above-mentioned incandescnet particle reaches pre-determined number, according to work as
When untill the detection number of above-mentioned incandescnet particle estimate that it is pre- prerequisite that the number of the signal of the light from above-mentioned incandescnet particle reaches
Time required for fixed number.
5. according to the method described in claim 1, it is characterised in that
In a period of untill the number of the signal of the light from above-mentioned incandescnet particle reaches pre-determined number, by each rule
Fixed time interval repeats above-mentioned photo detection area moving step, above-mentioned smooth detecting step, above-mentioned sequential light intensity data
Generation step, above-mentioned smoothing step and above-mentioned incandescnet particle detecting step,
This method is further comprising the steps of：Corrected according to the detection number of the above-mentioned incandescnet particle untill at that time it is above-mentioned as defined in
Time interval.
6. the method according to any one of Claims 1 to 5, it is characterised in that
In above-mentioned photo detection area moving step, make the position of above-mentioned photo detection area to move than the diffusion of above-mentioned incandescnet particle
Dynamic fireballing speed movement.
7. a kind of light analytical equipment, comfortable sample solution is carried out using the optical system detection of confocal microscope or multiphoton microscope
In disperse and the incandescnet particle of random motion light, the light analytical equipment is characterised by, including：
Photo detection area move portion, it makes the photo detection area of above-mentioned optical system by changing the light path of above-mentioned optical system
Position moved in above-mentioned sample solution；
Optical detection part, makes the position of above-mentioned photo detection area be moved in above-mentioned sample solution while by above-mentioned optical detection part
Detect the light from above-mentioned photo detection area；
Sequential light intensity data generating unit, its according to the detected photogenerated from above-mentioned photo detection area in temporal sequence
Sequential light intensity data；
Smoothing processing portion, it is smoothed to above-mentioned sequential light intensity data, until can ignore that above-mentioned incandescnet particle institute
The light sent is within the small time untill the missing of issuable data value；And
Signal processing part, it will have big on the above-mentioned sequential light intensity data after above-mentioned smoothing processing portion smoothing processing
Cause curve, the luminous intensity of hanging mitriform to change with time and be detected as the signal of the light from each above-mentioned incandescnet particle one by one,
Wherein this substantially hangs the light intensity distributions in the above-mentioned photo detection area of curve reflection of mitriform and is directed to and one with being in
Parameter in the range of the corresponding signal imagination of incandescnet particle,
Wherein, repeat using above-mentioned photo detection area move portion above-mentioned optical system photo detection area position shifting
Dynamic, being given birth to the detection of the light from above-mentioned photo detection area, using above-mentioned sequential light intensity data using above-mentioned optical detection part
Generation into the sequential light intensity data in portion, the smoothing processing to above-mentioned sequential light intensity data using above-mentioned smoothing processing portion
And the detection of the signal to the light from above-mentioned incandescnet particle using above-mentioned signal processing part, until above-mentioned signal processing part
Untill the number of the signal of the detected light from above-mentioned incandescnet particle reaches pre-determined number, according to from above-mentioned
The number of the signal of the light of incandescnet particle reaches the time required for pre-determined number to determine in above-mentioned sample solution
The concentration of above-mentioned incandescnet particle.
8. light analytical equipment according to claim 7, it is characterised in that
Repeat the light inspection using the above-mentioned optical system of above-mentioned photo detection area move portion by each predetermined time interval
Survey the movement of the position in region, using above-mentioned optical detection part to the detection of light from above-mentioned photo detection area, using above-mentioned
The generation of the sequential light intensity data of sequential light intensity data generating unit, using above-mentioned smoothing processing portion to above-mentioned sequential light intensity
The detection of the smoothing processing of degrees of data and the signal to the light from above-mentioned incandescnet particle of the above-mentioned signal processing part of utilization.
9. light analytical equipment according to claim 7, it is characterised in that
The concentration of the above-mentioned incandescnet particle in above-mentioned sample solution, the detection are determined according to the detection speed of above-mentioned incandescnet particle
Speed is the time according to required for the number of the signal of the light from above-mentioned incandescnet particle reaches pre-determined number and true
Fixed.
10. light analytical equipment according to claim 7, it is characterised in that also including with lower unit：
In a period of the unit is reached in the number of the signal of the light from above-mentioned incandescnet particle untill pre-determined number, root
Estimate that the number of the signal of the light from above-mentioned incandescnet particle reaches according to the detection number of the above-mentioned incandescnet particle untill at that time
Time required for pre-determined number.
11. light analytical equipment according to claim 7, it is characterised in that
In a period of untill the number of the signal of the light from above-mentioned incandescnet particle reaches pre-determined number, by each rule
Fixed time interval repeats the position of the photo detection area of the above-mentioned optical system using above-mentioned photo detection area move portion
Movement, using above-mentioned optical detection part to the detection of the light from above-mentioned photo detection area, utilize above-mentioned sequential luminous intensity number
According to the generation of the sequential light intensity data of generating unit, using above-mentioned smoothing processing portion to the smooth of above-mentioned sequential light intensity data
The detection of the signal to the light from above-mentioned incandescnet particle of above-mentioned signal processing part is handled and utilizes,
The light analytical equipment also include being corrected according to the detection number of the above-mentioned incandescnet particle untill at that time it is above-mentioned as defined in when
Between the unit that is spaced.
12. light analytical equipment according to claim 7, it is characterised in that
Also there is incandescnet particle to detect number display part, incandescnet particle detection number display part is shown in from above-mentioned incandescnet particle
The detection of the signal of light start after as the light from above-mentioned incandescnet particle detected by above-mentioned signal processing part signal
Number.
13. light analytical equipment according to claim 7, it is characterised in that
Also there is measurement end time display part, measurement end time display part display is according to from above-mentioned incandescnet particle
The detection of the signal of light start after as the light from above-mentioned incandescnet particle detected by above-mentioned signal processing part signal
Number and estimate, the number of the signal of light from above-mentioned incandescnet particle reach pre-determined number untill time.
14. the light analytical equipment according to any one of claim 7~13, it is characterised in that
Above-mentioned photo detection area move portion makes the position of above-mentioned photo detection area with the diffusion translational speed than above-mentioned incandescnet particle
Fast speed movement.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
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| JP2011009496 | 2011-01-20 | ||
| JP2011-009496 | 2011-01-20 | ||
| PCT/JP2012/051175 WO2012099234A1 (en) | 2011-01-20 | 2012-01-20 | Photoanalysis method and photoanalysis device using detection of light from single light-emitting particle |
Publications (2)
| Publication Number | Publication Date |
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| CN103328955A CN103328955A (en) | 2013-09-25 |
| CN103328955B true CN103328955B (en) | 2017-10-20 |
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| US (1) | US9863806B2 (en) |
| EP (1) | EP2667183A4 (en) |
| JP (1) | JP5856983B2 (en) |
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| WO2013157283A1 (en) * | 2012-04-18 | 2013-10-24 | オリンパス株式会社 | Method for detecting target particles |
| KR102126032B1 (en) * | 2013-07-31 | 2020-07-08 | 삼성전자주식회사 | Multi-channel fluorescence detecting module and nucleic acid analysis system having the same |
| CN105593667A (en) * | 2013-10-07 | 2016-05-18 | 奥林巴斯株式会社 | Photometric analysis device employing single light-emitting particle detection, photometric analysis method, and computer program for photometric analysis |
| JP6157326B2 (en) * | 2013-11-11 | 2017-07-05 | オリンパス株式会社 | Single luminescent particle detection method using light detection |
| JP6228858B2 (en) * | 2014-02-06 | 2017-11-08 | 日本電子株式会社 | Particle analysis apparatus and program |
| JP7029903B2 (en) * | 2017-08-09 | 2022-03-04 | シスメックス株式会社 | Sample processing equipment, sample processing system, and measurement time calculation method |
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Also Published As
| Publication number | Publication date |
|---|---|
| EP2667183A4 (en) | 2017-05-10 |
| US9863806B2 (en) | 2018-01-09 |
| WO2012099234A1 (en) | 2012-07-26 |
| US20130302906A1 (en) | 2013-11-14 |
| JP5856983B2 (en) | 2016-02-10 |
| CN103328955A (en) | 2013-09-25 |
| EP2667183A1 (en) | 2013-11-27 |
| JPWO2012099234A1 (en) | 2014-06-30 |
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